Field of the Invention
The disclosure relates to the field of microfluidic membrane sensing technology and more particularly, to methods and apparatus for nanomembrane based nucleic acid sensing platform.
Description of the Related Art
Dengue virus (DENV) is the world's fastest spreading tropical disease with an estimated 390 million people infected annually—three times more than the current estimate by WHO (Bhatt 2013, Mangold and Reynolds 2013). In the past several years, DENV has caused localized outbreaks in the continental U.S. (Jordan 2013). Some 3.6 billion people—about 40% of world population—are now at risk from DENV. Over two million people develop severe DENV, a serious condition requiring intensive hospital-based care, marked by internal hemorrhage (Gubler 2012, Kyle and Harris 2008). In the absence of a vaccine or any specific drug for its treatment, an early diagnosis enables appropriate supportive care that reduces disease associated morbidity and mortality (Tantawichien 2012). In addition, early diagnosis during the viremic period, which coincides with the first few days of onset of symptoms, can alert a physician to be especially watchful for warning signs of hemorrhage, which may require immediate hospital admission. Accurate diagnosis also reduces unnecessary prescription of antimalarials or antibiotics, thus reducing global drug resistance.
In the early stages of DENV infection, high levels of viremia are detected in the blood as compared to urine or saliva of the infected patient (Poloni 2010). Early diagnosis of patient's blood can thus be used as an early warning tool to identify regions of high prevalence, and institute public health measures such as insecticide spraying to control mosquitoes responsible for spreading of the disease.
While virus isolation remains the gold standard method of detecting the presence of viral pathogens, within the past couple of decades RT-PCR and ELISA-based methods have emerged as reliable diagnostic tools (Kao 2005, Lampman 2006, Lambert 2003, 2005, Pabbaraju 2009, Linssen 2000, Schmitt 2007, Saxena 2009, Weidmann 2010, Niyas 2010). However, these traditional methods of viral detection have a number of disadvantages that limit their usefulness for field applications. Results from virus isolation may take days and thus offer little actionable information during the course of an infection. ELISA techniques, although not time-consuming, typically requires 4-7 days for the body to produce appreciable antibodies by immunological response after virus infection and thus cannot be used as an early diagnostic marker. New assays are being developed which detect dengue nonstructural (NS-1) protein during acute viremia but the efficacy of these assays remains to be seen. Although the FDA recently cleared the first molecular test (CDC DENV-1-4 RT-PCR Assay) for dengue detection, the Applied Biosystems 7500 Fast Dx Real-time PCR Instrument is not portable and thus precious time is spent transporting specimens (2-3 hours of test time+potentially 24-48 hours of sample delivery time) and waiting for the results to return. A 2-3 hour result is a significant improvement but the complexity of such systems including its lack of portability, expensive reagents and requirement of technical personnel for operation become an economic disadvantage.
According to the FDA, two main groups of nucleic acid based diagnostic tests have been approved. The first group is for human genes and the other group is microbial tests. There are over 30 tests for human genetic detection, including, for example, cystic fibrosis, breast cancer, and prostate cancer. There are over 100 tests for microbial testing, including, for example, testing for E. coli, hepatitis, and enterococcus. In general, the tests use one of two specific detection assays. The first assay is called FISH (fluorescent in situ hybridization). The second assay consists of a few steps including some treatments of the sample, which usually means separation of nucleic acids from real samples (or raw samples), followed by PCR amplification, and subsequent optical detection, such as fluorescence detection. Each of these steps usually involves highly skilled investigators. Often a medical doctor is required to analyze and interpret the test results.
While there is abundant literature on DNA sensing technologies, little is reported about RNA detection. Among the RNA sensing technologies, most of the technologies rely on fluorescence-labeling for optical detection such as molecular beacon (Marti 2007), Förster resonance energy transfer (Sando and Kool 2002, Socher 2008) and dye-trapping liposomes (Baeumner 2004). Some optical detection techniques, for instance surface plasmon resonance imaging (SPRi) microarray (Nelson 2002) and mass-sensitive detection such as microcantilever designs (Zhang 2006) are label free, but require expensive equipment and have signal instability issues. Although these optical detection methods provide reasonable detection limits down to an impressive single molecule level, they all require costly, bulky optical systems.
Several label-free molecular sensing technologies like electrochemical sensors that amplify signal using redox reporters enhance detection sensitivity, yet are hampered by instability of the electrochemical signal and difficulty in calibration (Bakker and Qin 2006, Umezawa and Aoki 2004). Capacitance, conductance and field effect transistor (FET) electrode sensors are typically insensitive as the ionic strength within the electrical Debye layer on the electrode is about 2 to 3 times higher than the bulk and the presence of the RNA molecules would not significantly affect the local conductance (Stern 2007, Suni 2008). They are also expensive since they require fabrication of microelectrodes. Most importantly, the largest drawback of all electrode sensors is their long assay time due to diffusion-based transport of large target molecules like DNA/RNA to the electrode surface (Nkodo 2001). Several techniques have been suggested to remove the slow transport of large nucleic acid molecules to the electrode sensor. One involves the activation of high voltage at the electrode sensor to electrophoretically attract nearby DNAs (Sosnowski 1997). However, this electrophoretic concentration technique is highly non-specific and the elevated voltage can produce undesirable Faradaic reactions for high ionic strength buffers resulting in false current or voltage signals.
Therefore, the major disadvantages of existing detection technologies are 1) time consuming, 2) expensive, 3) require trained personnel and 4) not suitable for field diagnostics. Thus, the lack of availability of a point-of-care diagnostic platform has made the detection of viruses in human sera particularly cumbersome in endemic areas.